# Patent

## Title
Plants resistant to cucumber mosaic virus strain V34

## Abstract

The CP gene of CMV strain V34 (CMV-V34) is provided.

## Background
This application is divisional of U.S. Ser. No. 09/616,567, filed Jul. 14, 2000, which is now U.S. Pat. No. 6,342,655, which is a divisional o U.S. Ser. No. 08/875,233, filed Sep. 29, 1997, which is a now U.S. Pat. No. 6,127,601, which is a 371 of PCT/US95/07234 filed Jun. 7, 1995 which is a continuation of U.S. Ser. No. 08/367,789, filed Dec. 30, 1994, now abandoned.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to coat protein (CP) genes derived from WT strains of cucumber mosaic virus (CMV). More specifically, the invention relates to the genetic engineering of plants and to a method for conferring viral resistance to a plant using an expression cassette encoding CP genes of WT strains of CMV.
2. Description of the Prior Art
Many agriculturally important crops are susceptible to infection by plant viruses, particularly CMV, which can seriously damage a crop, reduce its economic value to the grower, and increase its cost to the consumer. Attempts to control or prevent infection of a crop by a plant virus such as CMV have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the genetic material which provides the protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. “Resistant” is the opposite of “susceptible,” and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
CMV is a single-stranded (+) ribonucleic acid (RNA) plant virus that has a functionally divided genome. The virus genome contains four RNA species designated RNAs 1-4. RNs 3 and 4 encode the coat protein (CP) which is a protein that surrounds the viral RNA and protects the viral RNA from being degraded. Only RNAs 1-3 are required for infectivity because the CP, which is encoded by RNA 4, is also encoded by RNA 3.
Several strains of CMV have been classified using serology, host range, peptide mapping, nucleic acid hybridization, and sequencing analyses. These CMV strains can be divided into two groups, which are designated “WT” (also known as subgroup I) and “S” (also known as subgroup II). The S group consists of at least three members. The WT group is known to contain at least 17 members.
Expression of the CP genes from tobacco mosaic virus, alfalfa mosaic virus, CMV, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus. Heterologous protection can also occur. For example, the expression of CP genes from watermelon mosaic virus-2 (WMV-2) or zucchini yellow mosaic virus (ZYMV) in transgenic tobacco plants has been shown to confer protection against six other potyviruses: bean yellow mosaic virus, potato virus Y, pea mosaic virus, clover yellow vein virus, pepper mottle virus, and tobacco etch virus. However, expression of a preselected CP gene does not reliably confer heterologous protection to a plant. For example, transgenic squash plants containing the CMV-C CP gene, a WT virus, which have been shown to be resistant to the CMV-C strain are not protected to the same degree against several other, highly virulent WT strains of CMV.
Thus, a need exists for plants resistant to WT strains of CMV.
SUMMARY OF THE INVENTION
This invention provides: an isolated and purified deoxyribonucleic acid (DNA) molecule that encodes the CP for the V27 strain of CMV (CMV-V27), and a chimeric expression cassette comprising this DNA molecule; an isolated and purified DNA molecule that encodes the CP for the V33 strain of CMV (CMV-V33), and a chimeric expression cassette comprising this DNA molecule; and an isolated and purified DNA molecule that encodes the CP for the V34 strain of CMV (CMV-V34), and a chimeric expression cassette comprising this DNA molecule; and an isolated and purified DNA molecule that encodes the CP for the A35 strain of CMV (CMV-A35), and a chimeric expression cassette comprising the DNA molecule. Another embodiment of the invention is exemplified by the insertion of multiple virus gene expression cassettes into one purified DNA molecule, e.g., a plasmid. Each of these cassettes also includes a promoter which functions in plant cells to cause the production of an RNA molecule, and at least one polyadenylation signal comprising 3′ nontranslated DNA which functions in plant cells to cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3′ end of the transcribed messenger RNA (mRNA) sequences, wherein the promoter is operably linked to the DNA molecule, and the DNA molecule is operably linked to the polyadenylation signal. Preferably, these cassettes include the promoter of the 35S gene of cauliflower mosaic virus (CaMV-35S gene) and the polyadenylation signal of the CaMV-35S gene (CaMV-35S).
Also provided are bacterial cells, and transformed plant cells, containing the chimeric expression cassettes comprising the CP genes derived from the CMV-V27, CMV-V33, CMV-V34, or CMV-A35 strains, and preferably the 35S promoter and the polyadenylation signal of the CaMV-35S gene. Plants are also provided, wherein the plants comprise a plurality of transformed cells containing the chimeric CP gene expression cassettes derived from the CMV-V27, CMV-V33, CMV-V34, or CMV-A35 strains, and preferably the promoter and the polyadenylation signal of the CaMV gene. Transformed plants of this invention include tobacco, beets, corn, cucumber, peppers, potatoes, melons, soybean, squash, and tomatoes. Especially preferred are members of the Cucurbitaceae (e.g., squash and cucumber,) and Solanaceae (e.g., peppers and tomatoes) family.
Another aspect of the present invention is a method of preparing a CMV-resistant plant, such as a dicot, comprising: transforming plant cells with a chimeric expression cassette comprising a promoter functional in plant cells operably linked to a DNA molecule that encodes a CP of a WT strain of CMV, e.g., V27, V33, V34, or A35; regenerating the plant cells to provide a differentiated plant; and identifying a transformed plant that expresses the CMV CP at a level sufficient to render the plant resistant to infection by the specific strains of CMV disclosed herein.
As used herein, with respect to a DNA molecule or “gene,” the phrase “isolated and purified” is defined to mean that the molecule is ether extracted from its context in the viral genome by chemical means and purified and/or modified to the extent that it can be introduced into the present vectors in the appropriate orientation, i.e., sense or antisense. As used herein, the term “chimeric” refers to the linkage of two or more DNA molecules which are derived from different sources, strains or species (e.g., from bacteria and plants), or the linkage of two or more DNA molecules, which are derived from the same species and which are linked in a way that does not occur in the native genome. As used herein, “expression” is defined to mean transcription or transcription followed by translation of a particular DNA molecule.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A. The nucleotide sequence of the CP gene of CMV-V27 (hereinafter “CMV-V27 CP”) from nucleotide position 1 to 360 (SEQ ID NO:1). The deduced amino acid sequence (SEQ ID NO:2) of the encoded open reading frame is shown below the nucleotide sequence.
FIG. 1B. The nucleotide sequence of the CP gene of CMV-V27 from nucleotide position 361 to 772 (SEQ ID NO:1). The deduced amino acid sequence (SEQ ID NO:2) of the encoded open reading frame is shown below the nucleotide sequence.
FIG. 2A. The nucleotide sequence of the CP gene of CMV-V33 (hereinafter “CMV-V33 CP”) from nucleotide position 1 to 420 (SEQ ID NO:3). The deduced amino acid sequence (SEQ ID NO:4) of the encoded open reading frame is shown below the nucleotide sequence.
FIG. 2B. The nucleotide sequence of the CP gene of CMV-V33 from nucleotide position 421 to 773 (SEQ ID NO:3). The deduced amino acid sequence (SEQ ID NO:4) of the encoded open reading frame is shown below the nucleotide sequence.
FIG. 3. The nucleotide sequence of the CP gene of CMV-V34 (hereinafter “CMV-34 CP”) from nucleotide position 1 to 771 (SEQ ID NO:5). The deduced amino acid sequence (SEQ ID NO:6) of the encoded open reading frame is shown below the nucleotide sequence.
FIG. 4A. The alignment of the nucleotide sequences of the CP genes from five CMV strains from nucleotide position 1 to 600. CMV-C CP (SEQ ID NO: 9) and CMV-WL CP (SEQ ID NO: 11) are described in Quemada et al. (J. Gen. Viral. 70:1065 1989)). The nucleotide sequence of CMV-V27 CP is shown in SEQ ID NO: 1, CMV-V33 CP is SEQ ID NO: 3 and CMV-V34 CP is SEQ ID NO: 5. Alignments were obtained with the use of the UWGCG Pileup program. The dots represent either the lack of sequence information at the 5′ end of the CP gene or gaps in homology in sequences relative to others in the alignment. The position of primer RMM351 is shown (SEQ ID NO: 7).
FIG. 4B. The alignment of the nucleotide sequences of the CP genes from five CMV strains (SEQ ID NOS: 1, 3, 5, 9 and 11) described in FIG. 4A from nucleotide position 601 to 840.
FIG. 4C. The alignment of the nucleotide sequences of the CP genes from five CMV strains (SEQ ID NOS: 1, 3, 5, 9 and 11) described in FIG. 4A from nucleotide position 841 to 1080.
FIG. 4D. The alignment of the nucleotide sequences of the CP genes from five CMV strains (SEQ ID NOS: 1, 3, 5, 9 and 11) from nucleotide position 1081 to 1170 alignment. The position of primer RMM352 is shown (SEQ ID NO: 8).
FIG. 5A. The alignment of the sequences of amino acid 1-150 deduced from the nucleotide sequences of CMV strains CMV-V27 CP (SEQ ID NO: 2), CMV-V33 CP (SEQ ID NO: 4), CMV-V34 CP (SEQ ID NO: 6), CMV-C CP (SEQ ID NO: 10) (shown in FIG. 4) and CMV strain Cmvq3 (Quemada et al., J. Gen. Virol. 70:1065 (1989)) (SEQ ID NO: 13) (hereinafter referred to as “CMV-Q3 CP”). The amino acid sequence of CMV-WL CP is shown in SEQ ID NO: 12. Alignments were performed by the UWGCG Pileup program. Differences among the WT virus strains are underlined and highlighted with asterisks. The dots represent gaps in homology in sequences relative to others in the alignment.
FIG. 5B. The alignment of the sequences of amino acid 151-219 deduced from the nucleotide sequences of CMV strains as described in FIG. 5A. (SEQ ID NOS: 2, 4, 6, 10, 12 and 13).
FIG. 6A. Assembly of CMV-V27 CP expression cassette. Polymerase chain reaction (PCR) products of CMV-V27 were installed into pCRII and subsequently inserted into pUC18cpexpress by routine methods. The bolded lines and arrows which are a part of the circle represent CaMV-35S sequences.
FIG. 6B. (FIG. 6A, continued.) Insertion of a CMV-V27 CP expression cassette BamHI fragment into the BglII site of pEPG204 and pEPG205 to produce pEPG239 and pEPG240, respectively.
FIG. 6C. Restriction map of pEPG239. This binary plasmid includes the CP expression cassettes for PRV (melon, long), CMV-V27 CP, ZYMV CP, and WMVII CP. For further information on PRV CP genes, refer to Applicants' International Patent Application No. PCT/US95/07272 entitled “Papaya Ringspot Virus Coat Protein Gene” filed on Jun. 7, 1995, incorporated by reference herein. For further information on ZYMV and WMV-2 CP genes, refer to Applicants' International Patent Application No. PCT/US89/03094 filed on Jul. 20, 1989 entitled “Potyvirus Coat Protein Genes and Plants Transformed Therewith”, incorporated by reference herein.
FIG. 6D. Restriction map of pEPG240. This binary plasmid includes the CP expression cassettes for PRV (melon, short), CMV-V27 CP, ZYMV CP, and WMVII CP.
FIG. 7A. Assembly of CMV-V33 CP expression cassette. PRC products of CMV-V33 CP were installed into pUC1318cpexpress by routine methods.
FIG. 7B. (FIG. 7A, continued.) Insertion of a CMV-V33 CP expression cassette BamHI fragment into the BglII site of pEPG204 and pEPG205 to produce pEPG196 and pEPG197, respectively.
FIG. 7C. Restriction map of pEPG196. This binary plasmid includes the CP expression cassettes for PRV (melon, long), CMV-V33 CP, ZYMV CP, and WMVII. Arrows indicate CaMV-35S promoter fragments.
FIG. 7D. Restriction map of pEPG197. This binary plasmid includes the CP expression cassettes for PRV (melon, short), CMV-V33 CP, ZYMV CP, and WMVII CP.
FIG. 8. The nucleotide sequence of the CP gene of CMV-A35 (SEQ ID NO: 14) (hereinafter “CMV-A35 CP). The deduced amino acid sequence of the encoded open reading frame is shown below the nucleotide sequence (SEQ ID NOS: 15-19).
FIG. 9A. The alignment of the amino acid sequences deduced from the nucleotide sequences of the six CMV strains shown in FIG. 10A for amino acid 1-120. (The majority is shown in SEQ ID NO: 20, CMV-C CP AA SEQ is SEQ ID NO: 10, CMV-C-A35 CP is SEQ ID NO: 15, CMV-V27 CP AA SEQ is SEQ ID NO: 2, CMV-V33 CP AA SEQ is SEQ ID NO: 4, CMV-V34 CP AA SEQ is SEQ ID NO: 6, and CMV-WL CP AA SEQ is SEQ ID NO: 12). Differences among the coat proteins are enclosed in boxes. The dashes represent gaps in homology in sequences relative to others in the alignment.
FIG. 9B. The alignment of the amino acid sequences deduced from the nucleotide sequences of the six CMV strains shown in FIG. 10 for amino acid 121 to 220 (SEQ ID NOS: 2, 4, 6, 10, 12 and 15).
FIG. 10A. The alignment of the nucleotide sequences of the CP genes from 6 CMV strains from nucleotide position 321-400 of a consensus sequence The majority is shown in SEQ ID NO: 21, CMV C-A35 CP is SEQ ID NO: 14, CMV-C CP is SEQ ID NO: 9, New CMV-V34 CP is SEQ ID NO: 5, CMV-WL CP is SEQ ID NO. 11, CMV-V27 CP is SEQ ID NO: 1 and CMV-V33 CP is SEQ ID NO: 3.). The dots represent either the lack of sequence information at the 5′ end of the CP gene or gaps in homology in sequences relative to others in the alignment.
FIG. 10B. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 401 to 480 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10C. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 481 to 560 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10D. The alignment of nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 561 to 640 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10E. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 641 to 720 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10F. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 721 to 800 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10G. The alignment of he nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 801 to 880 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10H. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 881 to 960 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10I. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 961 to 1040 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10J. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 1041 to 1120 (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).
FIG. 10K. The alignment of the nucleotide sequences of the CP genes of CMV strains described in FIG. 10A from nucleotide position 1121 to 1200. The dots represent gaps in homology in sequences relative to others in the alignment (SEQ ID NOS: 1, 3, 5, 9, 11, 14 and 21).



DETAILED DESCRIPTION OF THE INVENTION
The genome of CMV contains four RNA species designated RNA 1, 2, 3 and 4; 3389 nucleotides (nt), 3035 nt, 2193 nt, and 1027 nt, respectively (Peden et al., Virol., 53:487 (1973); Gould et al., Eur. J. Biochem., 126:217 (1982); Rezaian et al., Eur. J. Biochem., 143:227 (1984); Rezaian et al., Eur. J. Biochem. 150:331 (1985)). Only RNA 1, 2 and 3 are required for infectivity (Peden et al., Virol., 53:487 (1973)) because the CP, which is encoded by RNA 4, is also encoded by RNA 3. Translation of CMV RNA yield a 95 kiloDalton (kD) polypeptide from RNA 1, a 94 kD polypeptide from RNA 2 (Gordon et al., Virol., 123:284 (1983)), and two polypeptides from RNA 3: its 5′ end encodes a 35 kD polypeptide, and its 3′ end encodes a 24.5 kD polypeptide (Gould et al., Eur. J. Biochem., 126:217 (1982)). The 24.5 kD polypeptide is identical to that encoded by RNA 4 and is the CP.
Several strains of CMV have been classified using serology, host range, peptide mapping, nucleic acid hybridization, and sequencing. These CMV strains include two groups, WT and S. CMV WT strains include CMV-C, CMV-V27, CMV-V33, CMV-V34, CMV-M, CMV-O, CMV-Y, and CMV-A35 while S strains include CMV-Q, CMV-WL, and CMV-LS (Zaitlin et al., Viral., 201:200 (1994)). Protection against a strain in one group does not necessarily provide protection against all strains in that group. For example, transgenic squash plants protected with CP genes from the CMV-C are not protected against the CMV strains V27, V33, V34, or A35. In addition, Zaitlin et al. (Virol., 201:200 (1994)) report that tobacco plants transgenic for a CMV-FNY replicase gene show protection against challenge from WT strains but show no protection against challenge from S strain challenges. Thus, the present invention is directed to providing plants with resistance to WT strains of CMV, e.g., V27, V33, V34, or A35.
To practice the present invention, a viral gene must be isolated from the viral genome and inserted into a vector. Thus, the present invention provides isolated and purified DNA molecules that encode the CP of the V27, V33, or V34 strains of CMV. As used herein, a DNA molecule that encodes a CP gene includes nucleotides of the coding strand, also referred to as the “sense” strand, as well as nucleotides of the noncoding strand, complementary strand, also referred to as the “antisense” strand, either alone or in their base-paired configuration. Thus, a DNA molecule that encodes the CP of the V27 strain of CMV, for example, includes the DNA molecule having the nucleotide sequence of FIG. 1, a DNA molecule complementary to the nucleotide sequence of FIG. 1, as well as a DNA molecule which also encodes a CMV CP and its complement which hybridizes with a CMV-V27-specific DNA probe in hybridization buffer with 6×SSC, 5× Denhardt's reagent, 0.5% SDS and 100 micrograms per milliliter (μg/ml) denatured, fragmented salmon sperm DNA and remains bound when washed at 68° in 0.1×SSC and 0.5% SDS (Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989)). Moreover, the DNA molecules of the present invention can include non-CMV CP nucleotides that do not interfere with expression of the CMV CP gene. Preferably, the isolated and purified DNA molecules of the present invention comprise a single coding region for the CP. Thus, preferably the DNA molecules of the present invention are those consisting essentially of DNA that encodes the CP.
These CMV genes are used to produce the CPs, which are believed to confer resistance to viruses. Another molecular strategy to provide virus resistance in transgenic plants is based on antisense RNA. As is well known, a cell manufactures protein by transcribing the DNA of the gene encoding that protein to produce RNA, which is then processed to mRNA (e.g., by the removal of introns) and finally translated by ribosomes into protein. This process may be inhibited in the cell by the presence of antisense RNA. The term antisense RNA means an RNA sequence which is complementary to a sequence of bases in the mRNA in question in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3′ to 5′ sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5′ to 3′ sense. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, thus preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. This antisense RNA may be produced in the cell by transformation of the cell with an appropriate DNA construct arranged to transcribe the non-template strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).
The use of antisense RNA to downregulate the expression of specific plant genes is well known. Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference, e.g., lack of anthocyanin production in flower petals of petunia leading to colorless instead of colored petals (van der Krol et al., Nature, 333:866-869 (1988)); or at a more subtle biochemical level, e.g., change in the amount of polygalacturonase and reduction in depolymerization of pectin during tomato fruit ripening (Smith et al., Nature, 334:724-726 (1988)).
Another more recently described method of inhibiting gene expression in transgenic plants is the use of sense RNA transcribed from an exogenous template to downregulate the expression of specific plant genes (Jorgensen, Keystone Symposium “Improved Crop and Plant Products through Biotechnology”, Abstract X1-022 (1994)). Thus, both antisense and sense RNA have been proven to be useful in achieving downregulation of gene expression in plants, which are encompassed by the present invention.
The CMV CP gene does not contain the signals necessary for its expression once transferred and integrated into a plant genome. Accordingly, a vector must be constructed to provide the regulatory sequences such that they will be functional upon inserting a desired gene. When the expression vector/insert construct is assembled, it is used to transform plant cells which are then used to regenerate plants. These transgenic plants carry the viral gene in the expression vector/insert construct. The gene is expressed in the plant and increased resistance to viral infection is conferred thereby.
Several different methods exist to isolate a viral gene. To do so, one having ordinary skill in the art can use information about the genomic organization of cucumoviruses to locate and isolate the CP gene. The CP gene is located near the 3′ end of RNA 3. Using methods well known in the art, a quantity of virus is grown and harvested. The viral RNA is then separated by gel electrophoresis. A cDNA library is created using the viral RNA, by methods known to the art. The viral RNA is incubated with primers that hybridize to the viral RNA and reverse transcriptase, and a complementary DNA molecule is produced. A DNA complement of the complementary DNA molecule is produced and that sequence represents a DNA copy (cDNA) of the original viral RNA molecule. The DNA complement can be produced in a manner that results in a single double stranded cDNA or PCR can be used to amplify the DNA encoding the cDNA with the use of oligomer primers specific for viral sequences. These primers can include novel restriction sites used in subsequent cloning steps. Thus, a double stranded DNA molecule is generated which contains the sequence information of the viral RNA. These DNA molecules can be cloned in E. coli plasmid vectors after the additions of restriction enzyme linker molecules by DNA ligase. The various fragments are inserted into cloning vectors, such as well-characterized plasmids, which are then used to transform E. coli and create a cDNA library. CMV CP genes from previously isolated strains can be used as hybridization probes to screen the cDNA library to determine if any of the transformed bacteria contain DNA fragments with sequences coding for a CMV CP. Alternatively, plasmids which harbor CMV CP sequences can be determined by restriction enzyme digestion of plasmids in bacterial transformants. The cDNA inserts in any bacterial colonies which contain this region can be sequenced. The CP gene is present in its entirety in colonies which have sequences that extend 5′ to the sequence which encodes the ATG start codon and sequences that extend 3′ of the stop codon.
Alternatively, cDNA fragments can be inserted in the sense orientation into expression vectors. Antibodies against the CP can be used to screen the cDNA expression library and the gene can be isolated from colonies which express the protein.
In the present invention, the DNA molecules encoding the CP genes of the CMV strains V27, V33, V34, and A35 have been determined and the genes have been inserted into expression cassettes. These expression cassettes can be individually placed into a vector that can be transmitted into plants, preferably a binary vector. Alternatively, two or more of the CMV CP genes can each be present in an expression cassette which can be placed into the same binary vector, or any of the CMV CP expression cassettes of the present invention can be placed into a binary vector with one or more viral gene expression cassettes. The expression vectors contain the necessary genetic regulatory sequences for expression of an inserted gene. The CP gene is inserted such that those regulatory sequences are functional and the genes can be expressed when incorporated into a plant genome. For example, vectors of the present invention can contain combinations of expression cassettes that include DNA from a heterologous CMV CP gene (i.e., one from another CMV isolate), papaya ringspot virus (PRV) CP gene, a ZYMV CP gene, and a WMV-2 CP gene.
Moreover, when combinations of viral gene expression cassettes are placed in the same binary plasmid, and that multigene cassette containing plasmid transformed into a plant, the viral genes all preferably exhibit substantially the same degrees of efficacy when present in transgenic plants. For example, if one examines numerous transgenic lines containing two different intact viral gene cassettes, the transgenic line will be immune to infection by both viruses. Similarly, if a line exhibits a delay in symptom development to one virus, it will also exhibit a delay in symptom development to the second virus. Finally, if a line is susceptible to one of the viruses it will be susceptible to the other. This phenomenon is unexpected. If there were not a correlation between the efficacy of each gene in these multiple gene constructs this approach as a tool in plant breeding would probably be prohibitively difficult to use. Even with single gene constructs, one must test numerous transgenic plant lines to find one that displays the appropriate level of efficacy. The probability of finding a line with useful levels of expression can range from 10-50% (depending on the species involved). For further information refer to Applicants' International Patent Application No. PCT/US95/06261 entitled “Transgenic Plants Expressing DNA Constructs Containing a Plurality of Genes to Impart Virus Resistance” filed on Jun. 7, 1995, incorporated by reference herein.
In order to express the viral gene, the necessary genetic regulatory sequences must be provided. In the present invention, the CP genes are inserted into vectors which contain cloning sites for insertion 3′ of the initiation codon and 5′ of the poly(A) signal. The promoter is 5′ of the initiation codon such that when genes are inserted at the cloning site, a functional unit is formed in which the inserted genes are expressed under the control of the various genetic regulatory sequences.
The segment of DNA referred to as the promoter is responsible for the regulation of the transcription of DNA into mRNA. A number of promoters which function in plant cells are known in the art and can be employed in the practice of the present invention. These promoters can be obtained from a variety of sources such as plant or plant viruses, and can include, but are not limited to, promoters isolated from the caulimovirus group such as the CaMV-35S promoter (CaMV-35S), the enhanced CaMV-35S promoter (enh-CaMV-35S), the figwort mosaic virus full-length transcript promoter FMV-34S), and the promoter isolated from the chlorophyll a/b binding protein. Other useful promoters include promoters which are capable of expressing the cucumovirus proteins in an inducible manner or in a tissue-specific manner in certain cell types in which the infection is known to occur. For example, the inducible promoters from phenylalanine ammonia lyase, chalcone synthase, hydroxyproline rich glycoprotein, extensin, pathogenesis-related proteins (e.g. PR-1a), and wound-inducible protease inhibitor from potato may be useful.
Preferred promoters for use in the present CP-containing cassettes include the constitutive promoters from CaMV, the tumor-inducing (Ti) genes nopaline synthase (NOS) (Bevan et al., Nucleic Acids Res., 11:369 (1983)) and octopine synthase (Depicker et al., J. Mol. Appl. Genet., 1:561 (1982)), and the bean storage protein gene phaseolin. The poly(A) addition signals from these genes are also suitable for use in the present cassettes. The particular promoter selected is preferably capable of causing sufficient expression of the DNA coding sequences to which it is operably linked, to result in the production of RNA or proteins effective to provide viral resistance, but not so much as to be detrimental to the cell in which they are expressed. The promoters selected should be capable of functioning in tissues including, but not limited to, epidermal, vascular, and mesophyll tissues. The actual choice of the promoter is not critical, as long as it has sufficient transcriptional activity of their respective RNAs to accomplish the expression of the preselected proteins and their subsequent conferral of viral resistance to the plants.
The nontranslated leader sequence can be derived from any suitable source and can be specifically modified to increase the translation of the mRNA. The 5′ nontranslated region can be obtained from the promoter selected to express the gene, an unrelated promoter, the native leader sequence of the gene or coding region to be expressed, viral RNAs, suitable eucaryotic genes, or a synthetic gene sequence. The present invention is not limited to the constructs presented in the following examples.
The termination region or 3′ nontranslated region which is employed is one which will cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3′ end of the transcribed mRNA sequence. The termination region can be native with the promoter region, native with the gene, or can be derived from another source, and preferably include a terminator and a sequence coding for polyadenylation. Suitable 3′ nontranslated regions of the chimeric plant gene include but are not limited to: (1) the 3′ transcribed, nontranslated regions containing the polyadenylation signal of Agrobacterium Ti plasmid genes, such as the NOS gene; and (2) plant genes like the soybean 7S storage protein genes.
Preferably, the expression cassettes of the present invention are engineered to contain a constitutive promoter 5′ to its translation initiation codon (ATG) and a poly(A) addition signal (AATAAA) 3′ to its translation termination codon. Several promoters which function in plants are available, however, the preferred promoter is the 35S constitutive promoters from CaMV. The poly (A) signal can be obtained from the CaMV-35S gene or from any number of well characterized plant genes, i.e., NOS, octopine synthase, and the bean storage protein gene phaseolin. The constructions are similar to that used for the expression of the CMV-C CP in PCT Patent Application PCT/US88/04321, published on Jun. 29, 1989 as WO 89/05858, claiming the benefit of U.S. Ser. No. 135,591, filed Dec. 21, 1987, entitled “Cucumber Mosaic Virus Coat Protein Gene”, and the CMV WL CP in PCT Patent Application PCT/US89/03288, published on Mar. 8, 1990 as WO 90/02185, claiming the benefit of U.S. Ser. No. 234,404, filed Aug. 19, 1988, entitled “Cucumber Mosaic Virus Coat Protein Gene.”
Selectable marker genes can be incorporated into the present expression cassettes and used to select for those cells or plants which have become transformed. The marker gene employed may express resistance to an antibiotic, such as kanamycin, gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Other markers could be employed in addition to or in the alternative, such as, for example, a gene coding for herbicide tolerance such as tolerance to glyphosate, sulfonylurea, phosphinothricin, or bromoxynil. Additional means of selection could include resistance to methotrexate, heavy metals, complementation providing prototrophy to an auxotrophic host, and the like.
The particular marker employed will be one which will allow for the selection of transformed cells as opposed to those cells which are not transformed. Depending on the number of different host species one or more markers can be employed, where different conditions of selection would be useful to select the different host, and would be known to those of skill in the art. A screenable marker such as the β-glucuronidase gene can be used in place of, or with, a selectable marker. Cells transformed with this gene can be identified by the production of a blue product on treatment with 5-bromo-4-chloro-3-indoyl-β-D-glucuronide.
In developing the present expression construct, i.e., expression cassette, the various components of the expression construct such as the DNA molecules, linkers, or fragments thereof will normally be inserted into a convenient cloning vector, such as a plasmid or phage, which is capable of replication in a bacterial host, such as E. coli. Numerous cloning vectors exist that have been described in the literature. After each cloning, the cloning vector can be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, resection, insertion, in vitro mutagenesis, addition of polylinker fragments, and the like, in order to provide a vector which will meet a particular need.
For Agrobacterium-mediated transformation, the expression cassette will be included in a vector, and flanked by fragments of the Agrobacterium Ti or root-inducing (Ri) plasmid, representing the right and, optionally the left, borders of the Ti or Ri plasmid transferred DNA (T-DNA). This facilitates integration of the present chimeric DNA sequences into the genome of the host plant cell. This vector will also contain sequences that facilitate replication of the plasmid in Agrobacterium cells, as well as in E. coli cells.
All DNA manipulations are typically carried out in E. coli cells, and the final plasmid bearing the cucumovirus expression cassette is moved into Agrobacterium cells by direct DNA transformation, conjugation, and the like. These Agrobacterium cells will contain a second plasmid, also derived from Ti or Ri plasmids. This second plasmid will carry all the vir genes required for transfer of the foreign DNA into plant cells. Suitable plant transformation cloning vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as generally disclosed in Glassman et al. (U.S. Pat. No. 5,258,300), or Agrobacterium rhizogenes. 
A variety of techniques are available for the introduction of the genetic material into or transformation of the plant cell host. However, the particular manner of introduction of the plant vector into the host is not critical to the practice of the present invention, and any method which provides for efficient transformation can be employed. In addition to transformation using plant transformation vectors derived from the Ti or Ri plasmids of Agrobacterium, alternative methods could be used to insert the DNA constructs of the present invention into plant cells. Such methods may include, for example, the use of liposomes, electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA, 82:824 (1984)), chemicals that increase the free uptake of DNA (Paszkowski et al., EMBO J., 3:2717 (1984)), DNA delivery via microprojectile bombardment (Klein et al., Nature, 327:70 (1987)), microinjection (Crossway et al., Mol. Gen. Genet., 202:179 (1985)), and transformation using viruses or pollen.
The choice of plant tissue source or cultured plant cells for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is regenerable, in that it will retain the ability to regenerate whole, fertile plants following transformation.
The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA carrying the present viral gene expression cassette(s) for an effective period of time. This can range from a less-than-one-second pulse of electricity for electroporation, to a two-to-three day co-cultivation in the presence of plasmid-bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet Corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.
Following treatment with DNA, the plant cells or tissue may be cultivated for varying lengths of time prior to selection, or may be immediately exposed to a selective agent such as those described hereinabove. Protocols involving exposure to Agrobacterium will also include an agent inhibitory to the growth of the Agrobacteriun cells. Commonly used compounds are antibiotics such as cefotaxime and carbenicillin The media used in the selection may be formulated to maintain transformed callus or suspension culture cells in an undifferentiated state, or to allow production of shoots from callus, leaf or stem segments, tuber disks, and the like.
Cells or callus observed to be growing in the presence of normally inhibitory concentrations of the selective agents are presumed to be transformed and may be subcultured several additional times on the same medium to remove nonresistant sections. The cells or calli can then be assayed for the presence of the viral gene cassette, or can be subjected to known plant regeneration protocols. In protocols involving the direct production of shoots, those shoots appearing on the selective media are presumed to be transformed and can be excised and rooted, either on selective medium suitable for the production of roots, or by simply dipping the excised shoot in an Ri compound and directly planting it in vermiculite.
In order to produce transgenic plants exhibiting viral resistance, the viral genes must be taken up into the plant cell and stably integrated within the plant genome. Plant cells and tissues selected for their resistance to an inhibitory agent are presumed to have acquired the selectable marker gene encoding this resistance during the transformation treatment. Since the marker gene is commonly linked to the viral genes, it can be assumed that the viral genes have similarly been acquired. Southern blot hybridization analysis using a probe specific to the viral genes can then be used to confirm that the foreign genes have been taken up and integrated into the genome of the plant cell. This technique may also give some indication of the number of copies of the gene that have been incorporated. Successful transcription of the foreign gene into mRNA can likewise be assayed using Northern blot hybridization analysis of total cellular RNA and/or cellular RNA that has been enriched in a polyadenylated region. mRNA molecules encompassed within the scope of the invention are those which contain viral specific sequences derived from the viral genes present in the transformed vector which are of the same polarity as that of the viral genomic RNA such that they are capable of base pairing with viral specific RNA of the opposite polarity to that of viral genomic RNA under conditions described in Chapter 7 of Sambrook et al. (1989). Moreover, mRNA molecules encompassed within the scope of the invention are those which contain viral specific sequences derived from the viral genes present in the transformed vector which are of the opposite polarity as that of the viral genomic RNA such that they are capable of base pairing with viral genomic RNA under conditions described in Chapter 7 in Sambrook et al. (1989).
The presence of a viral gene can also be detected by immunological assays, such as the double-antibody sandwich assays described by Namba et al., Gene, 107:181 (1991) as modified by Clark et al., J. Gen. Virol., 34:475 (1979). See also, Namba et al., Phytopathology, 82:940 (1992). Cucumovirus resistance can also be assayed via infectivity studies as generally disclosed by Namba et al., ibid., wherein plants are scored as symptomatic when any inoculated leaf shows vein clearing, mosaic or necrotic symptoms.
Seed from plants regenerated from tissue culture is grown in the field and self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines which are evaluated for viral resistance in the field under a range of environmental conditions. The commercial value of viral-resistant plants is greatest if many different hybrid combinations with resistance are available for sale. The farmer typically grows more than one kind of hybrid based on such differences as maturity, color or other agronomic traits. Additionally, hybrids adapted to one part of a country are not adapted to another part because of differences in such traits as maturity, disease and insect tolerance. Because of this, it is necessary to breed viral resistance into a large number of parental lines so that many hybrid combinations can be produced.
The invention will be further described by reference to the following detailed examples. Enzymes were obtained from commercial sources and were used according to the vendor's recommendations or other variations known in the art. Other reagents, buffers, etc., were obtained from commercial sources, such as Sigma Chemical Co., St. Louis, Mo., unless otherwise specified.
Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail in, for example, in European Patent Application Publication Number 223,452, published Nov. 29, 1986, which is incorporated herein by reference. General references containing such standard techniques include the following: R. Wu, ed., METHODS IN ENZYMOLOGY, Vol. 68 (1979); J. H. Miller, EXPERIMENTS IN MOLECULAR GENETICS (1972); J. Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); and D. M. Glover, ed , DNA CLONING VOL. II (1982).
FIGS. 6 and 7 are presented to illustrate the constructions of this invention.
EXAMPLE 1
A. Isolation of CMV RNAs
Zucchini squash plants (20-day old) were inoculated with CMV strains V27, V33, or V34; after 7-10 days, infected leaves were harvested and CMV virus particles were isolated. The procedure used was based on IBM protocols from Lot et al., Annals of Phytopathology, 4:25 (1972), Francki et al., CMI/AAB DESCRIPTIONS OF PLANT VIRUSES, (July, 1979), and Habili and Francki, Virology, 57:292 (1974). Approximately 100 grams (g) of fresh leaves were extracted in an equal weight per volume (w/v) of 0.5 molar (M) Na-citrate (pH 6.5) containing 5 millimolars (mM) EDTA and 100 milliliters (ml) of chloroform. After centrifugation of the extract at 12,000×G for 10 minutes, polyethyleneglycol (“PEG”, Sigma Chemical Co. PEG-8000, average molecular weight, Research Grade) was added to the supernatant to a final concentration of 10% and the suspension was stirred for 30-40 minutes at 0-4° C. This suspension was centrifuged at 12,000×G for 10 minutes, and the pellet was resuspended in 40-50 ml of 5 mM Na-borate buffer (pH 9.0) containing 0.5 M EDTA. TRITON X-100 was then added to the virus particle suspension to a final concentration of 2% and stirred on ice for 30 minutes. This suspension was then centrifuged at 19,000×G for 15 minutes, and the supernatant was collected and subsequently centrifuged at 105,000×G for 2 hours. The virus pellet was collected and resuspended in about 2 ml of 5 mM Na-borate buffer (pH 9.0) containing 0.5 mM EDTA. The resuspended virus preparation was applied onto a step sucrose gradient consisting of 5 layers: 5%, 10%, 15%, 20%, and 25% sucrose dissolved in 2.0 mM Na-phosphate buffer (pH 7.5). Gradients were centrifuged at 37,000 rpm in a Sorvall TH641 swinging bucket rotor for 45 minutes. After centrifugation, the virus band was harvested, the virus preparation was dialyzed against Na-borate buffer, and LiCl was added (2 M final concentration) to lyse the virions and to precipitate viral RNA. CMV RNA was dissolved and reprecipitated with ethanol and dissolved in water. By agarose gel electrophoresis, the expected four RNA species were observed.
B. Cloning CMV Coat Protein Genes
(a) CMV-V27
The first cDNA strand of CMV-V27 was synthesized with the use of Perkin-Elmer RT-PCR kit reagents and the primer RMM352 (shown in FIG. 4); immediately in the same reaction tube, a PCR was carried out with the use of oligonucleotide primers RMM351 and RMM352 (shown in FIG. 4), following the manufacturer's protocol. The ATG translation start is included in the NcoI site present in primer RMM351. Individual PCR product molecules were cloned using the TA CloningTM kit (Invitrogen Corp., San Diego, Calif.) into pCRII (included in the TA CloningTM kit as a linearized plasmid with single 3′ dT overhangs at the ends of the molecule). Three clones were isolated for further study: CMVV27TA21, CMVV27TA23, and CMVV27TA26. With the use of a kit (Sequenase 2 purchased from USB, Cleveland, Ohio), the CMV-V27 insert in clone CMVV27TA21 was sequenced.
The coat protein sequence of CMV-V27 was compared to the coat protein sequence from 12 different CMV isolates: CMV-BAUL, CMV-Q3, CMV-WL, CMV-TRK7, CMV-FC, CMV-IL7F, CMV-C, CMV-PR50, CMV-P6, CMV-O, CMV-M and CMV-Y. CMV-V27 CP is similar to CMV-Y CP in that it contains a serine at position 29 while other strains have an alanine at this position. However, CMV-Y CP contains a leucine at position 18 while CMV-V27 CP contains a proline at position 18. In addition, CMV-V27 CP has a methionine at position 206, no other CMV coat proteins have a methionine at this position (Baulcombe, D., “Mutational analysis of CMV RNA3: Effects on RNA3 accumulation, RNA4 synthesis and plant infection.” Unpublished Direct Submission. Submitted (19 Jun. 1992) David Baulcombe, The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, NR2 7UH, United Kingdom; Hayakawa et al., Gene, 71:107 (1988); Hayakawa et al., J. Gen. Virol. 70:499 (1989); Owen et al., J. Gen. Virol., 71:2243 (1990); Pappu et al., “The nucleotide and the deduced amino acid sequences of CP genes of three Puerto Rican isolates of CMV.” Unpublished (1992). This sequence is included in the Geneank sequence data base; Salanki et al., “Complete nucleotide sequence of RNA 3 from CMV strain Trk 7.” Unpublished (1993). This sequence is included in the GeneBank data base; Shintaku, J. Gen. Virol. 72:2587 (1991)).
(b) CMV-V33
CMV-V33 was purified and viral RNA extracted from a virion preparation as described above; subsequently single stranded cDNA was synthesized using Perkin-Elmer RT-PCR kit reagents and oligomer primer RMM352. The CP gene of strain V33 was amplified using PCR as described above for V27 with the use of oligomer primers RMM351 and RMM352 (FIG. 4). The V33 CP gene PCR product was digested with NcoI and directly cloned into the expression cassette cpexpress installed into pUC1318 (see Kay and McPherson, Nucleic Acids Research, 15:2779 (1987) for pUC1318; Slightom, Gene, 100:251 (1991) for cpexpress; pUC1318cpexpress is the cpexpress described in Slightom, however it is installed into the HindIII site of the modified pUC plasmid pUC1318 described in detail in Kay and McPherson), rather than into the intermediate vector pCRII. By colony hybridization with a CMV CP probe, a number of clones were identified for further analysis: V33cel, V33ce2, V33ce7, and V33ce9. The CMV-V33 insert in clone V33ce7 was sequenced with the use of a kit (Sequenase 2 purchased from USB, Cleveland, Ohio).
The coat protein sequence of CMV-V33 was compared to the coat protein sequence from 13 different CMV isolates: CMV-BAUL, CMV-Q3, CMV-WL, CMV-TRK7, CMV-FC, CMV-IL7F, CMV-C, CMV-PR50, CMV-V27, CMV-P6, CMV-O, CMV-M and CMV-Y, CMV-V33 CP has a serine at position 67 while all other CMV strains compared included a proline at this position. At position 196, both CMV-V33 CP and CMV-Y CP have a valine residue; all other CMV isolates contains isoleucine at this position. However, at position 184, CMV-V33 CP has an alanine residue while CMV-Y has a threonine residue. Therefore, CMV-V33 CP is unique (Baulcombe, D., “Mutational analysis of CMV RNA3: Effects on RNA3 accumulation, RNA4 synthesis and plant infection.” Unpublished Direct Submission. Submitted (19 Jun. 1992) David Baulcombe, The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, NR2 7UH, United Kingdom; Hayakawa et al., Gene, 71:107(1988); Hayakawa et al., J. Gen. Virol. 70:499 (1989); Owen et al., J. Gen. Virol., 1:2243(1990); Pappu et al., “The nucleotide and the deduced amino acid sequences of coat protein genes of three Puerto Rican isolates of cucumber mosaic virus.” Unpublished (1992). This sequence is included in the GeneBank sequence data base; Salanki et al., “Complete nucleotide sequence of RNA 3 from cucumber mosaic virus strain Trk 7.” Unpublished (1993). This sequence is included in the GeneBank data base; Shintaku, J. Gen. Virol. 72:2587 (1991)).
(c) CMV-V34
CMV-V34 RNA was prepared as described above. Subsequently, the first cDNA strand was synthesized using CMV-V34 template in a reaction that included the following: approximately 2 μg CMV-V34 RNA, 1×buffer for Superscript Reverse Transcriptase (supplied by BRLGIBCO, Grand Island, N.Y.), 2 mM dNTPs, oligomer primer RMM352 (37.5 μg/ml), 1.5 microliters (μl) RNasin, and 1 Al Superscript Reverse Transcriptase (BRL-GIBCO) in a 20-μl reaction. After this reaction was allowed to proceed for 30 minutes, an aliquot of the first strand reaction was used as a template in a PCR to amplify the CMV-V34 CP gene. The CMV-V34 CP gene PCR product was cloned into the pCRII vector included in the TA Cloning' Kit supplied by InvitrogenCorp. Two clones were isolated for further study: TA17V34 and TA112V34. The CMV-V34 insert of clone TA17V34 was sequenced with the use of a kit (Sequenase 2 purchased from USB, Cleveland, Ohio). Comparative sequence analysis of the CMV-V34 CP gene with other CMV CP genes CMV-BAUL., CMV-Q3, CMV-WL, CMV-TRK7, CMV-FC, CMV-IL7F, CMV-C, CMV-PR50, CMV-V27, CMV-P6, CMV-O, CMV-M and CMV-Y showed that the CMV-V34 CP gene is unique (Baulcombe, D. Mutational analysis of CMV RNA3: Effects on RNA3 accumulation, RNA4 synthesis and plant infection. Unpublished Direct Submission. Submitted (19 Jun. 1992) David Baulcombe, The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, NR2 7UH, United Kingdom; Hayakawa et al., Gene, 71:107 (1988); Hayakawa et al., J. Gen. Virol. 70:499 (1989); Owen et al., J. Gen. Virol., 71:2243 (1990); Pappu et al., (1992) “The nucleotide and the deduced amino acid sequences of coat protein genes of three Puerto Rican isolates of cucumber mosaic virus.” Unpublished. This sequence is included in the GeneBank sequence data base; Salanki et al., “Complete nucleotide sequence of RNA 3 from cucumber mosaic virus strain Trk 7.” Unpublished (1993) This sequence is included in the GeneBank data base; Shintaku, J. Gen. Virol. 72:2587 (1991)).
C. Engineering CMV CP Genes
(a) CMV-V27
The NcoI fragment in CMVV27TA21 that harbors CMV-V27 CP coding sequences was excised from CMVV27TA21 and inserted into the plant expression cassette cpexpress in pUC18 to give CMVV27TA21ce42. The resulting expression cassette was isolated as a partial HindIII fragment and inserted into the binary vector pGA482G [The parent binary plasmid was pGA482, constructed by An (Plant Physiol., 81:86 (1986)). This binary vector contains the T-DNA border sequences from pTiT37, the selectable marker gene NOS-NPTII (which contains the plant-expressible nopaline gene promoter fused to the bacterial NPTII gene obtained from Tn5), a multiple cloning region, and the cohesive ends of phage lambda (An, Plant Physiol., 81:86 (1986))] to yield pEPG191 and pEPG192. Subsequently, a PRV CP expression cassette was installed to obtain a binary vector that included both CMV-V27 CP and PRV CP expression cassettes.
Alternatively, the CMV-V27 CP Ncol fragment obtained from CMVV27TA21 was installed into pUC1318cpexpress (see Kay et al., Nucleic Acids Research, 15:2779 (1987) for pUC1318; Slightom, Gene 100:251 (1991) for cpexpress; pUC1318cpexpress is the cpexpress described in Slightom, however it is installed into the HindIII site of the modified pUC plasmid pUC 1318 described in detail in Kay et al.) to give the plasmid CMVV27TA210E13 (similar to CMVV27TA21ce42). The plasmid pUC1318 provided additional sites (e.g., BamHI and Xbal) with which the cassette could be inserted into the binary vector pGA482G. Subsequently, the bacteria-derived gentamicin-(3)-N-acetyl-transferase gene (Allmansberger et al., Mol. Gen. Genet., 198:514 (1985)) was installed into a Sall site outside of the T-DNA region, adjacent to the left border (BL)). The BamHI fragment harboring the CMV-V27 CP expression cassette was isolated and inserted into the Bglll site of the binary plasmid pEPG205 (PRV34/Z72/WMBN22) to give pEPG240 (CMVV27/PRV34/Z72/WMBN22). The BamHI fragment was also installed into the BglII site of the binary plasmid pEPG204 (PRV16/Z72/WMBN22) to yield pEPG239 (CMVV2716/PRV16/Z72/WMBN22) (Table 1). For further information on PRV CP genes, refer to Applicants' International Patent Application No. PCT/US95/07272 entitled “Papaya Ringspot Virus Coat Protein Gene” filed on Jun. 7, 1995, incorporated by reference herein. For further information on ZYMV and WMV-2 CP genes, refer to Applicants' International Patent Application No. PCT/US89/03094 filed on Jul. 20, 1989 entitled “Potyvirus Coat Protein Genes and Plants Transformed Therewith”, incorporated by reference herein.











TABLE 1





Binary
Parental Plasmid
Site
CMVcn Cassette
pEPG#







pGA482G
pGA482G
Hindi=
CMVV27cpexpress
191 or






192


pPRBN
pEPG204(P16sZW)
BglII
CMVV27cpexpress
239


pPRBN
pEPG2045
BglII
CMVV27cpexpress
240



(P16sZW)


pPRBN
pEPG106(ZW)
Hindi=
CMVV27cpexpress
243


pGA482G
pGA482G
Hindi=
CMVV33ce7
198


pPRBN
pEPG106(ZW)
Hindi=
CMVV33ce7
244


pPRBN
pEPG204(P16sZW)
BglII
CMVV33ce7
196


pPRBN
pEPG205(P34sZW)
BglII
CMVV33ce7
197


pGA482G
pGA482G
Hindi=
17V34cpexp113
190









(b) CMV-V33
Subsequently, both HindIII and BamHI fragments were excised from clone V33ce7; these fragments carried the complete expression cassette for CMV-V33 CP gene. The BamHI fragment (V33 CP expression cassette) was inserted into the BglII site of pEPG204 PRV16/ZY72MMBN22) to obtain pEPG196. The BamHI fragment was also inserted into the BglII site of pPEPG205 (PRV34/ZY72/WMBN22) to obtain pEPG197 (V3329/PRV34/ZY72/WMBN22). The HindIII fragment harboring the V33 CP cassette was installed into pGA482G to obtain pEPG198 (Table 1).
(c) CMV-V34
An NcoI fragment excised from clone TA17V34 was installed into the NcoI site of pUC1318 cpexpress. A resulting plasmid that includes the CMV-V34 coding NcoI fragment inserted in the sense orientation is 17V34/cpexp113. A partial HindIII fragment from the plasmid 17V34/cpexp113 was isolated and installed into pGA482G to yield pEPG190 (Table 1).
(d) Agrobacterium Strains
The binary plasmids described here, such as pPRBN (for further information on these plasmids, refer to Applicants' International Patent Application No. PCT/US95/06261 entitled “Transgenic Plants Expressing DNA Constructs Containing a Plurality of Genes to Impart Virus Resistance” filed on Jun. 7, 1995, incorporated by reference herein) or their derivatives, can be transferred into Agrobacterium strains A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRi278b), Mog301 and others. Strains A208, C58, LBA4404, and A4RS are available from ATCC, 12301 Parklawn Drive, Rockville, Md. A4RS (pRi278b) was obtained from Dr. F. Casse-Delbart, C.N.R.A., Route de Saint Cyr, F78000, Versailles, France. C58Z707 was obtained from Dr. A. G. Hepburn, University of Illinois, Urbana, Ill. Mog301 was obtained from Mogen Nev., Leiden, Netherlands.
D. Transfer of CMV Coat Protein Genes to Tobacco
In order to test whether the CMV CP gene constructs described herein confcr protection against CMV challenge with homologous strains, some of the binary plasmids listed above (e.g., pEPG197, pEPG198, pEPG239, and pEPG240) have been used to insert these novel CMV CP genes into Nicotiana tabacum. Agrobacterium-mediated transfer of the plant expressible CMV CP genes described herein was done using the methods described in PCT published application WO 89/05859, entitled “Agrobacterium Mediated Transformation of Germinating Plant Seeds.
Five R1 progeny lines of Nicotiana t. transformed with 35 the binary plasmid pEPG239 and five R1 progeny lines of Nicotiana t. transformed with the binary plasmid pEPG240 have been obtained. These binary plasmids include the CP gene of CMV-V27. The ten Ro parental plants of these lines were assayed for NPTII protein expression by ELISA. They each expressed NPT II protein by ELISA. Furthermore, these ten lines were assayed for both the nptII and CMV-V27 CP genes by PCR analysis. PCR analysis detected both genes in all ten Ro plants.
The binary plasmid pEPG198 was used to obtain 11 R0 transgenic Nicotiana t. plants. By PCR analysis, the CMV-V33 CP gene was detected in nine of the eleven R0 plants tested.
E. Cloning and Engineering CMV-A35 PC Gene
20-day-old zucchini squash plants in the greenhouse were inoculated with CMV-A35; after 7-10 days infected leaves were harvested. Total RNA was isolated from these infected plants by the use of Tri-Reagent and the instructions provided with the reagent (Molecular Research Center, Inc., Cincinnati, Ohio). Single-stranded cDNA was synthesized using total RNA template. The reaction included 1× first Strand cDNA Synthesis Buffer (GIBCO-BRL), 1 mM dNTP's (Pharmacia), 2 μl oligonucleotide primer RMM352 (150 μg/ml), 2 μl RNasin (Promega), and 1 μl RTase Superscript II (GIBCO-BRL) in a 20 μl reaction volume. The CMV-A35 CP gene was PCR amplified with the use of CMV CP-specific primers RMM351 and 352. The PCR included 3 μl of the cDNA synthesis reaction described above, 8 μl of each primer RMM351 and RMM352 (150 μg/μl stock), 5 μl 10× reaction buffer, 4 μl dNTP's (10 mM), 1.5 μl MgCl2 (50 mM), and 0.5 μl, Taq polymerase (BRL-GIBCO). PCR conditions were carried out as follows: 93° 45 sec, 50° 45 sec, then 72° 180 sec for 30 cycles, then 72° for 5 min, then. hold at 4°. PCR products were visualized by agarose gel electrophoresis and subsequently cloned. PCR product molecules were cloned into the pCRII vector supplied with the TA cloning kit (Invitrogen Corp.) Four clones were identified and restriction mapped, however, none were sequenced for further analysis.
Alternatively, an aliquot of the CMV-A35 PCR product was digested with NcoI and ligated it into the NcoI site of pUC19B2 cpexpress to give the plasmid CMV-A35cpexp33. The CP insert of this plasmid was sequenced with the use of the Sequenase II Kit supplied by USBiochemical (FIG. 8). Sequence analysis reveals that CMV-A35 CP sequence differs from the CP sequences of CMV-C, CMV-V27, CMV-V33, CMV-V34, and CMV-WL (FIGS. 9 and 10). For example, A35 differs from the other CMV-C strains referred to above at amino acid position #26 (FIG. 9). Examination of the nucleotide sequence comparisons differs from other CMV CP genes characterized (FIG. 10).
A BamHI/BIlII fragment was excised from A35cpexp33 and installed into the unique BglII site pGA482G. The plasmid pUC19B2cpexp provides a BamHI site at the 5′ end of the cpexp cassette and a BglII site at the 3′ end of the expression cassette. Upon insertion into a BglII site, the unique BglII site of the binary plasmid pGA482 is maintained for subsequent insertions of gene cassettes. Binary plasmids that include the CMV-A35 expression cassette are being transformed into various Agrobacterium strains (e.g., C58Z707, Mog301, and LBA4404). These Agrobacterium strains are used to transform plants to impart resistance to CMV CARNA5.
All publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

## Claims

1. An isolated and purified DNA molecule comprising DNA encoding the coat protein of the V34 strain of cucumber mosaic virus.

2. The isolated and purified DNA molecule of claim 1 wherein the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:5.

3. A vector comprising a chimeric expression cassette comprising the DNA molecule of claim 1, a promoter and a polyadenylation signal, wherein the promoter is operably linked to the DNA molecule, and the DNA molecule is operably linked to the polyadenylation signal.

4. The vector of claim 3, wherein the promoter is the cauliflower mosaic virus 35S promoter.

5. The vector of claim 4 wherein the polyadenylation signal is the polyadenylation signal of the cauliflower mosaic 35S gene.

6. A bacterial cell comprising the vector of claim 3.

7. The bacterial cell of claim 6 wherein the bacterial cell is selected from the group consisting of an Agrobacterium tumefaciens and an Agrobacterium rhizogenes cell.

8. A transformed plant cell transformed with the vector of claim 3.

9. The transformed plant cell of claim 8 wherein the promoter is cauliflower mosaic virus 35S promoter and the polyadenylation signal is the polyadenylation signal of the cauliflower mosaic 35S gene.

10. A plant selected from the family Cucurbitaceae comprising a plurality of the transformed cells of claim 8.

11. A plant selected from the family Solanaceae comprising a plurality of the transformed cells of claim 8.

12. A method of preparing a cucumber mosaic viral resistant plant comprising:
transforming plant cells with a chimeric expression cassette comprising a promoter functional in plant cells operably inked to a DNA molecule that encodes a coat protein; wherein the DNA molecule is from cucumber mosaic virus strain V34; regenerating the plant cells to provide a differentiated plant; and 
identifying a transformed plant that expresses the cucumber mosaic virus coat protein at a level sufficient to render the plant resistant to infection by cucumber mosaic virus strain V34.

13. The method of claim 12 wherein the plant is a dicot.

14. The method of claim 13 wherein the dicot is selected from the family Cucurbitaceae.

15. The method of claim 13 wherein the dicot is selected from the family Solanaceae.

16. A vector comprising a chimeric expression cassette comprising the DNA molecule of claim 1 and at least one chimeric expression cassette comprising a heterologous CMV coat protein gene which is not the coat protein gene of the V34 strain of CMV, a papaya ringspot virus coat protein gene, a zucchini yellow mosaic virus coat protein gene, or a watermelon mosaic virus II coat protein gene, wherein each expression cassette comprises a promoter and a polyadenylation signal, wherein the promoter is operably linked to the DNA molecule or coat protein gene, and the DNA molecule or coat protein gene is operably linked to the polyadenylations signal.

17. A bacterial cell comprising the vector of claim 16.

18. A plant cell transformed with the vector of claim 16.

19. The plant cell of claim 18 wherein the promoter is cauliflower mosaic virus 35S promoter and the polyadenylation signal is the polyadenylation signal of the cauliflower mosaic 35S gene.

20. A plant selected from the family Cucurbitaceae comprising a plurality of the transformed cells of claim 18.

21. A plant selected from the family Solanaceae comprising a plurality of the transformed cells of claim 18.